G-Olig2 cells were designed by the insertion of green fluorescent protein (GFP) into the gene for Olig2, a lineage-specific transcription factor.This insertion permits visualization and physical separation of a subset of living ES-cell-derived neural cells.

G-Olig2 cells were designed by the insertion of green fluorescent protein (GFP) into the gene for Olig2, a lineage-specific transcription factor.

Storage Conditions

liquid nitrogen vapor phase

Derivation

SCRC-1037 is a subclone of a deposited cell line.

Comments

G-Olig2 cells were designed by the insertion of green fluorescent protein (GFP) into the gene for Olig2, a lineage-specific transcription factor. This insertion permits visualization and physical separation of a subset of living ES-cell-derived neural cells. [PubMed: 12529550] SCRC-1037 is a subclone of deposited cell line.

Note: To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.

Feeder Cell Preparation for Subcultures

Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.

One hour before subculturing the ES cells, perform a 100% medium change for the MEFs using complete growth medium for ES cells.

Add 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC® 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.

Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.

Spin the cells at 270 x g for 5 min. Aspirate the supernatant.

Resuspend in enough complete growth medium for ES cells to reseed new vessels at the desired split ratio (i.e. a split ratio of 1:4 to 1:7 is recommended). Perform a cell count to determine the total number of cells. ES cells should be plated at a density of 30,000 – 50,000 cells/ cm2.

Add separate aliquots of the cell suspension to the appropriate size flask containing feeder cells and add an appropriate volume of fresh complete growth medium for ES cells to each vessel.

Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1-2 days.

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In addition, prior to purchase, for-profit commercial institutions must obtain a license agreement from Washington University-St. Louis. For instructions on how to proceed, please contact ATCC's Office of Corporate Development at licensing@atcc.org.